Piceatannol Protects Against Age-Related Hearing Loss By Inhibiting Cellular Pyroptosis And Inflammation Through Regulated Caspase-11-GSDMD Pathway

Objective:To investigate the role of Caspase-11-GSDMD signaling pathway in age-related hearing loss in aged mice,and to investigate the protective effect mechanism of Piceatannol on age-related hearing loss,in order to provide an experimental basis for developing leucovorin as a safe and effective clinical drug for the prevention and treatment of hearing loss.Methods:1.For the efficacy study,mice were randomly divided into 5 groups: control,48-week-old geriatric,geriatric + PCT(2.5 mg/kg),geriatric + PCT(5 mg/kg)and geriatric+ PCT(10 mg/kg)groups.For the mechanistic study mice were randomly divided into four groups: control group,48-week-old geriatric group,geriatric + BAY11-7082(5 mg/kg)and geriatric + PCT(10 mg/kg)group,and PCT was dissolved in distilled water as the therapeutic drug.PCT was injected intraperitoneally once a day for 7 days.Auditory Brain Response(ABR)was detected in rats after completion of modeling;H&E staining was performed to detect histopathological damage to the cochlea;immunohistochemistry was performed to detect relevant protein expression;protein immunoblotting was performed to detect NLRP3,ASC,NF-KB,Caspase-1,Caspase-4/11,GSDMD IL-1β and IL-18.2.D-gal+ LPS model establishment: Human coronary artery endogenous cells(HEI-OC-1)cells were randomly divided into the following experimental groups:(1)Control;(2)D-gal+ LPS;(3)D-gal+ LPS + PCT-2.5 μM;(4)D-gal+ LPS + PCT-5μM;(5)D-gal+ LPS + To explore the effect of the inflammatory vesicle inhibitor BAY11-7082 on inflammatory damage in aging,we performed another experiment on the following groups:((1)Control;(2)D-gal+ LPS;(3)D-gal+ LPS + BAY11-7082(5 μM)and(4)D-gal+ LPS + PCT-10 μ.Subsequently,HEI-OC-1 was incubated at 30 mg/kg D-gal and 1 μg/m L LPS under co-incubation conditions for 48 h.Cell viability changes were detected by CCK-8;cell ROS levels were detected by flow cytometry as well as DCFH-DA probe;expression levels of key proteins were detected by cellular immunofluorescence;protein immunoblotting was performed to detect NLRP3,ASC,NF-KB,Caspase-1,Caspase-4/11,GSDMD,IL-1β and IL-18 protein expression levels by protein immunoblotting.Results:1.Hearing thresholds were significantly elevated in the aged group compared with the young group of mice.PCT treatment group could effectively alleviate the hearing loss.HE results showed that PCT could attenuate the hearing loss induced by accumulated inflammation in aging mice in vivo and histopathological damage of inner ear structure in mice was reduced compared with the young group.Immunohistochemical results indicated that the expression level of Caspase-11 and NLRP3 proteins in the cochlear tissue of mice in the aging group was obviously higher than that of mice in the younger group,and expression of Caspase-11 and NLRP3 in the cochlear tissue was inhibited after PCT treatment.Westren Blot revealed that,compared with the young group,changes in level of expression of inflammatory vesicles NLRP3,ASC,NF-KB and pyroptosis protein Caspase-11,Caspase-1,GSDMD,IL-1β and IL-18 protein expression were significantly increased in the old group compared to the young group,and all were downregulated to different degrees after PCT treatment.2.The results of CCK-8 assay showed that HEI-OC-1 cells showed decreased cell viability and increased cell damage in a simulated senescent inflammatory environment;PCT group could significantly increase cell viability as well as reduce damage.Flow cytometry as well as DCFH-DA probe results showed that PCT treatment group could significantly reduce intracellular ROS levels compared with the D-gal+LPS group;as observed by cellular immunofluorescence,PCT treatment group significantly reduced the protein fluorescence intensity of Caspase-4/11,NLRP3 and GSDMD compared with the D-gal+LPS group.Protein immunoblotting results showed that PCT inhibited the intracellular inflammation level.Moreover,in addition,Westren Blot results showed that the PCT group significantly reduced the expression of inflammatory vesicles NLRP3,ASC,NF-KB and pyroptosis proteins Caspase-11,Caspase-1,GSDMD-N,IL-1β,IL-18 proteins compared with the D-gal+ LPS group.Conclusion:The results of the present study suggest that PCT treatment can attenuate cochlear damage in ARHL mice,and the potential mechanism may be to reduce ARHL-induced inner ear structural damage by decreasing the occurrence of cellular inflammation to reduce pyroptosis.